Expression of Recombinant Stonustoxin Alpha Subunit and Preparation of polyclonal antiserum for Stonustoxin Neutralization Studies.

Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of Stonustoxin from Synanceia horrida and prepare antibodies against it The SNTXα gene sequence was optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 60 mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s serum exhibited a concentration of 1.5 mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7 ng. In the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom.

The Natterin Proteins Diversity: A Review on Phylogeny, Structure, and Immune Function.

Since the first record of the five founder members of the group of Natterin proteins in the venom of the medically significant fish Thalassophryne nattereri, new sequences have been identified in other species. In this work, we performed a detailed screening using available genome databases across a wide range of species to identify sequence members of the Natterin group, sequence similarities, conserved domains, and evolutionary relationships. The high-throughput tools have enabled us to dramatically expand the number of members within this group of proteins, which has a remote origin (around 400 million years ago) and is spread across Eukarya organisms, even in plants and primitive Agnathans jawless fish. Overall, the survey resulted in 331 species presenting Natterin-like proteins, mainly fish, and 859 putative genes. Besides fish, the groups with more species included in our analysis were insects and birds. The number and variety of annotations increased the knowledge of the obtained sequences in detail, such as the conserved motif AGIP in the pore-forming loop involved in the transmembrane barrel insertion, allowing us to classify them as important constituents of the innate immune defense system as effector molecules activating immune cells by interacting with conserved intracellular signaling mechanisms in the hosts.

Anaphylaxis induced by Thalassophryne nattereri venom in mice is an IgE/IgG1-mediated, IL-4-dependent phenomenon.

We hypothesized that beyond the Thalassophryne nattereri venoms ability to induce in mice a strong specific-Th2 response with high levels of specific IgE/IgG1, it would be able to trigger anaphylaxis in sensitized individuals. To investigate whether the venom is capable of inducing an allergic reaction in mice and characterize soluble and cellular mediators involved in this process, BALB/c female mice were sensitized intraperitoneally with decreasing-dose of venom at weekly intervals for 4 weeks and challenged by intraperitoneal, oral or epicutaneous routes with venom 2 weeks later. Our data show that sensitized-mice challenged by all routes showed intense symptoms of anaphylaxis, dependent on the anaphylactic IgG1 and IgE antibodies and mast cells. The late-phase reaction developed after initial symptoms was characterized by the influx of eosinophils, dependent on IL-5, IL-17A and eotaxin produced by Th2 cells in inflamed lungs and skin draining lymph-nodes. Using C57BL/6 deficient mice we demonstrated that IL-4 KO mice failed to develop anaphylactic symptoms or local Th2 inflammation, producing low levels of IgG1 and increased levels of IgG2a. Together our results demonstrated that the venom of T. nattereri has allergenic proteins that can trigger an allergic process, a phenomenon IgE-IgG1 dependent, IL-4-mediated and negatively regulated by IFN-γ.

Neutrophils releasing IL-17A into NETs are essential to plasma cell differentiation in inflamed tissue dependent on IL-1R.

Interleukin (IL) 17A in chronic inflammation is also produced by innate immune cells as neutrophils. Mice with chronic humoral response induced by venom of Thalassophryne nattereri (VTn) proved to be a good tool for evaluating the impact of IL-17A on the development of long-lived plasma cells in the inflamed peritoneal cavity. Here, we report that VTn induces IL-17A production by neutrophils accumulating in the peritoneal cavity and triggers the extrusion of IL-17A along with neutrophil extracellular traps (NETs). Neutrophil depletion reduced the number of IL17A-producing cells in VTn-immunized mice and blocked the differentiation of long-lived plasma cells. Specific antibody production and survival of long-lived plasma cells was ablated in VTn-immunized mice deficient in CD4, while CD28 signaling had the opposite effect on differentiation of long-lived plasma cells. Further, maturation of long-lived plasma cells in inflamed peritoneal cavity was IL-1R1 and COX-2 dependent. Finally, when both the Raf-MEK-ERK pathway and the IL-17A or IL-1R1 activities were blocked, neutrophils were unable to promote the differentiation of memory B cells into long-lived plasma cells, confirming the essential role of neutrophils and IL-17A along with NETs in an IL-1/IL-1R-dependent manner as the novel helping partner for plasma cell differentiation in chronically inflamed tissues.

The hierarchical process of differentiation of long-lived antibody-secreting cells is dependent on integrated signals derived from antigen and IL-17A.

Switched CD19-positive memory B cells purified from mice with chronic immune response against Thalassophrynenattereri venom proteins were cultured with venom or cytokines. Our results confirm the existence of a hierarchic process of differentiation: activated memory B cells progressively acquire increasing levels of CD138 and decreasing levels of CD45R/B220 to finally arrive at ASC with B220(neg) phenotype, which are IgG1-secreting cells. Only Bmem from peritoneal cavity or bone marrow of VTn immunized mice presented the capacity to generate ASC functionally active. IL-17A or IL-21/IL-23/IL-33 improves the ability of venom to induce intracellular IgG of peritoneal derived-ASC. Cognate stimulation with venom and IL-17A is sufficient to down-regulate the expression of CD45R/B220. BAFF-R is up-regulated in splenic or medullar derived-ASC stimulated by venom, CpG or cytokines. Only splenic derived-ASC up-regulate Bcl-2 expression after CpG or the combination of IL-21/IL-23/IL-33 stimulation. Finally, the activation of ASC for IgG1 secretion is triggered by venom proteins in peritoneal cavity and by IL-17A in medullar niche. These results show the importance of the integration of signals downstream of BCR and IL17-A receptors in modulating ASC differentiation, focusing in the microenvironment niche of their generation.

Neutralizing antibodies obtained in a persistent immune response are effective against deleterious effects induced by the Thalassophryne nattereri fish venom.

Thalassophryne nattereri envenoming represents a great cost to North and Northeast Brazilian communities in terms of public healths, leisure and tourism. Victims rapidally develop symptoms as pain, local swelling, erythema followed by intense necrosis that persist for long days. The aim of this work was tested the immune competence of neutralizing antibodies in pre-immunized mice against principal toxic activities induced by venom. During the primary antibody response in mice, an elevation of IgG antibody levels was only observed on day 28. After boosting, high antibody levels were detected between days 49 and 70, with a 12-fold increase in IgG level over control values at day 49. We confirmed the in vitro neutralizing capacity of T. nattereri anti-venom against toxic effects and thereafter we show that neutralizing antibodies obtained in a persistent immune response are more effective, inclusive against edematous reaction. After boosting during the secondary response mice with high antibody levels do not present any alterations in venule or arteriole after topical application of venom on cremaster muscle. In addition, CK activity diminished in these mice with high neutralizing antibody levels corroborating the attenuation of the myonecrotic effect by venom. In addition, we determined the presence of high IgG antibodies levels in patients 6 months after injury by T. nattereri. In conclusion, the presence of neutralizing antibodies against to T. nattereri venom in the serum of pre-immunized mice could change the outcome of lesion at site of posterior envenoming. Antigen-specific antibodies of high affinity in consequence to specific immune response, dependent of T lymphocyte activation, could minimize the symptoms of intense and immediate inflammatory reaction caused by T. nattereri venom. These finding prompt us to the possibility of development of immune therapeutic strategies using specific anti-venom as an efficient intervention for protecting human victims.

Analysis of the inflammatory reaction induced by the catfish (Cathorops spixii) venoms.

Cathorops spixii is one of the most abundant venomous fish of the southeastern coast of the State of São Paulo, and consequently causes a great part of the accidents seen there. The accidents affect mainly fishermen, swimmers and tourists and are characterized by punctiform or wide wounds, erythema, edema, pain, sudoresis, indisposition, fever, nausea, vomiting and secondary infection. The objective of this work was to characterize the inflammatory response induced in mice by both venoms (mucus and sting) of the catfish C. spixii. Our results demonstrated that both venoms induced a great number of rolling and adherent leukocytes in the post-capillary venules of cremaster muscle of mice, and an increase in the vascular permeability in peritoneal cavity. Mucus induced the recruitment of neutrophils immediately after injection followed later by macrophage infiltration. In contrast, the cellular infiltration elicited by sting venom was rapidly resolved. The peritonitis reaction provoked by venoms was characterized by cytokine (IL-6), chemokines (MCP-1 and KC) or lipid mediator (LTB4) production in the peritoneal cavity. The macrophages from 7-day mucus venom-induced exudates upon in vitro mucus venom stimulation, expressed CD11c x MHC class II and release bioactive IL-12p70. On the other hand, sting venom-elicited peritoneal macrophages lost the ability to differentiate into dendritic cells, following re-stimulation in vitro with sting venom, they do not express CD11c, nor do they exhibit sufficient levels of MHC class II. In conclusion, both types of venoms (mucus or sting) promote inflammatory reaction with different profiles, and the inflammatory reaction induced by the first was characterized by antigen persistence in peritoneal cavity that allowed the activation of phagocytic cells with capacity of antigenic presentation.

Experimental immunization with Thalassophryne nattereri fish venom: striking IL-5 production and impaired of B220+ cells.

Murine experimental model have been useful to understanding the toxic as well as the pharmacological properties of the Thalassophryne nattereri venom. However, the specific immune response to T. nattereri venom in mice is yet unclear. Our results showed that the venom elicited in BALB/c mice high levels of specific IgG1 and total IgE isotype with high affinity, accompanied by a striking IL-5 production, what point out to a Th2-like response. Meanwhile, the production of IFN-gamma by lymphocytes pool expanded upon mitogen stimulus, suggests that the venom was also able to activate Th1 clones. Elevated number of antigen-presenting cells expressing CD11c or CD11b from day 4 to 6 supported ongoing antigen presentation process in the primary response and efficient T-cell expansion (increase of CD4(+) cells). In contrast, decreased B220 expression was observed, suggesting that the formation of memory long lived cell compartment. In conclusion, T. natterri venom stimulates an association of cytokine of both Th1 and Th2 profile, with a notable IL-5 production and specific IgG1 and total IgE isotypes secretion. Furthermore, our finding showed that T. natterri venom can affect the B cell fate and induce a memory antibody response through the secretion of protective IgG subclasses. Further studies with the venom protein toxins may provide clues to molecular mechanism regulating proliferation and differentiation of antibody-secreting cells in our model. A better understating of how T. natterri venom can modulate immune response could be useful in therapeutic strategies.

Resposta imune induzida pelas peçonhas do bagre Cathorops agassizii / The immune response induced by venoms of Cathorops agassizii

A produção de toxinas por animais aquáticos é uma estratégia importante que garante sua sobrevivência em um ecossistema altamente competitivo. Constituem-se de uma rica fonte de agentes bioquímicos altamente ativos o que aumenta ainda mais a relevância das pesquisas nessa área. Nossos estudos objetivaram caracterizar as respostas imune inata e específica induzidas pelas peçonhas do muco e do ferrão do bagre Cathorops agassizii. A coleta dos espécimes foi realizada no complexo Baía-Estuário de Santos e São Vicente, localizado no litoral sul do Estado de São Paulo. As peçonhas (do Muco e do Ferrão) apresentaram perfil eletroforético similar entre si. Induzida a inflamação aguda em um modelo experimental murino, as peçonhas apresentaram igualmente a capacidade de induzir aumento da permeabilidade vascular e também edema de pata. A detecção de Leucotrieno B4 e Prostaglandina E2 no lavado da cavidade peritoneal dos camundongos injetados, com ambas as peçonhas, corroboram esta hipótese. Nossos resultados através da mícroscopia intravital mostraram que as peçonhas induzem um grande número de leucócitos rolantes nas vênulas pós-capilares com focos de extravasamento leucocitárío, principalmente de neutrófilos seguido pelo influxo de macrófagos. Além disso, a peçonha do Ferrão induziu uma resolução mais rápida do influxo leucocitário ao contrário da peçonha do Muco que manteve o infiltrado macrofágico por até 7 dias. De maneira interessante, somente a citocina IL -6 foi detectada no lavado peritoneal induzida principalmente pela peçonha do Muco e as quimiocinas KC e MCP-l, por ambas as peçonhas, expressando naquele momento, a participação destes mediadores no recrutamento de neutrófilos e macrófagos para o sítio da lesão. As peçonhas foram eficazes ao induzir uma produção primária e secundária de anticorpos das classes IgM e IgG anti-venenos...

Lethal, oedema, haemorrhagic activity of spotted butterfish (Scatophagus argus, Linn) sting extract and its neutralization by antiserum and pharmacological antagonists.

An attempt has been made in this communication to develop antiserum in rabbit against Scatophagus. argus sting extract. Antiserum did not neutralized the sting extract induced proinflammatory and haemorrhagic activity but successfully neutralized lethality upto 2LD50. Cyproheptadine, indomethacin and BW 755C pretreatment significantly reduced sting extract induced proinflammatory activity. The haemorrhagic activity of sting extract was significantly inhibited by temperature, UV-exposure, EDTA, cyproheptadine, indomethacin and BW 755C pretreatment. The results conclude that the local effects of S.argus venom is likely to be mediated through release of mediators and may be encountered by pharmacological antagonists better than the antiserum.

Adrenergic and cholinergic activity contributes to the cardiovascular effects of lionfish (Pterois volitans) venom.

The aim of the present study was to further investigate the cardiovascular activity of Pterois volitans crude venom. Venom (0.6-18 microg protein/ml) produced dose- and endothelium-dependent relaxation in porcine coronary arteries that was potentiated by atropine (10nM), but significantly attenuated by the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (NOLA; 0.1mM), by prior exposure of the tissue to stonefish antivenom (SFAV, 3 units/ml, 10 min), or by removal of extracellular Ca(2+). In rat paced left atria, venom (10 microg protein/ml) produced a decrease, followed by an increase, in contractile force. Atropine (0.5 microM) abolished the decrease in force and potentiated the increase. Propranolol (5 microM) did not affect the decrease in force but significantly attenuated the increase. In spontaneously beating right atria, venom (10 microg protein/ml) produced an increase in rate that was significantly attenuated by propranolol (5 microM). Prior incubation with SFAV (0.3 units/microg protein, 10 min) abolished both the inotropic and chronotropic responses to venom. In the anaesthetised rat, venom (100 micro protein/kg, i.v.) produced a pressor response, followed by a sustained depressor response. Atropine (1mg/kg, i.v.) potentiated the pressor response. The further addition of prazosin (50 microg/kg, i.v.) restored the original response to venom. Prior administration of SFAV (100 units/kg, i.v., 10 min) significantly attenuated the in vivo response to venom. It is concluded that P. volitans venom produces its cardiovascular effects primarily by acting on muscarinic cholinergic receptors and adrenoceptors. As SFAV neutralised many of the effects of P. volitans venom, we suggest that the two venoms share a similar component(s).

An investigation of the biological activity of bullrout (Notesthes robusta) venom.

Bullrout envenomation is known to cause intense pain. Crude bullrout venom and venom fractions were assessed for protease, hyaluronidase, phospholipase and hemolytic activities, reactivity with stonefish antivenom, lethality to brine shrimp and ability to elicit pain in human subjects. Compared with venom obtained from frozen specimens, live fish venom-milking techniques rendered greater venom potency and improved storage characteristics. Although mild proteolytic and hemolytic activity was observed, crude venom demonstrated no hyaluronidase or phospholipase A2 activity, did not affect brine shrimp, or show antigenicity with stonefish antivenom. A single venom protein isolated from bullrout venom is attributed with causing pain in human subjects. The sensations elicited by this novel algesic protein are consistent with chemical stimulation of polymodal nociceptors.

The effects of antivenom on the in vitro neurotoxicity of venoms from the taipans Oxyuranus scutellatus, Oxyuranus microlepidotus and Oxyuranus scutellatus canni.

The venoms of the inland (Oxyuranus microlepidotus), coastal (O. scutellatus) and Papuan (O. s. canni) taipans are among the most potent in the world. The present study compared the in vitro neurotoxic effects of these venoms and the protective effects of taipan antivenom. Venom (10 microg/ml) from all three snakes abolished nerve-mediated twitches of the chick biventer cervicis muscle preparation with the following rank order of potency (based on the time taken to inhibit 90% of the twitch response; t90): O. microlepidotus (27+/-3 min) > O. scutellatus (42+/-3 min) = O. S. canni (48+/-5 min). This inhibitory effect of all three venoms was primarily postsynaptic in origin as evidenced by the inhibition of responses to exogenous acetylcholine (ACh; 1 mM) and carbachol (CCh; 20 microM), but not potassium chloride (40 mM). In contrast, the presynaptic neurotoxins taipoxin (3 microg/ml) and paradoxin (3 microg/ml) abolished nerve-mediated twitches without producing a significant effect on contractile responses to exogenous agonists. Prior incubation of the tissue with taipan antivenom (1 unit/ml for 10 min) markedly attenuated the inhibitory effects of taipoxin (3 microg/ml) and paradoxin (3 microg/ml), as well as O. scutellatus (10 microg/ml) and O. s. canni (10 microg/ml) venom. However, in the presence of antivenom, O. microlepidotus venom (10 microg/ml) still abolished nerve-mediated twitches and responses to ACh and CCh. The results of the current study indicate that taipan antivenom, raised against O. scutellatus venom, is effective, in vitro, against the neurotoxic effects of venom from the Papuan and coastal taipans, as well as the presynaptic effects of venom from the inland taipan. However, the antivenom appears less effective against the postsynaptic effects of the latter. It is possible that inland taipan venom contains a component not neutralised by the antivenom which may contribute to the extreme potency of this venom.

Production and characterization of monoclonal antibodies against stonustoxin from Synanceja horrida.

Stonustoxin (SNTX), a lethal factor purified from the venom of stonefish Synanceja horrida, is a protein (148,000 mol. wt) existing as a dimer comprising two subunits (alpha and beta) of mol. wts 71,000 and 79,000, respectively. Its LD50 (i.v.) is 17 ng/g in mice and it causes haemolysis of rat and rabbit erythrocytes in vitro. Eight monoclonal antibodies (Mabs) against SNTX have been developed using the Balb/C mouse. These Mabs have been purified by Protein G affinity membrane disc chromatography. They were all classified as IgG1 with half of them having kappa and the rest lambda light chains. They had affinity constants ranging from 3.75 x 10(-9) to 9.74 x 10(-9) M. Six were able to protect mice from a challenge of a lethal dose of SNTX. However, not all protective Mabs were able to neutralize the haemolytic effect in vitro. Only four Mabs (31A, 32B, 38A and 46A) could inhibit rat and rabbit erythrocyte haemolysis, while one Mab (43D) offered partial inhibition and another Mab (8A) did not inhibit haemolysis at all. The non-protective Mabs (43B and 44G) were also incapable of neutralizing haemolysis. Five epitopes were recognized by the eight Mabs. Four Mabs (31A, 32B, 38A and 46A) were found to have similar epitope specificity while the rest were directed at different epitopes on the SNTX molecule. Thus these results suggest that the domain on the SNTX molecule responsible for lethality is probably distinct from the domain important for in vitro haemolytic activity.

Experimental myasthenia gravis is inhibited by receptor-antireceptor complexes.

Experimental autoimmune myasthenia gravis (EAMG) induced in rabbits by immunisation with purified nicotinic acetylcholine receptor from Torpedo marmorata is a highly reproducible model for the human disease. Pretreatment of experimental animals with immune complexes containing receptor and anti-receptor antibodies suppressed the subsequent induction of EAMG. Animals were protected from the normal severe paralysis. Moreover, antibody levels were reduced by synthesis of antibody rapidly terminated. Possible mechanisms are discussed.